This invention relates to a method of making ultra-thin dense skin gas-controlling asymmetrical membranes and compositions containing ultra-thin dense skin gas-controlling asymmetrical membranes.
The growth and culturing of biological material such as cells, tissues, and organs is essential to the scientific and health care fields. World wide demand for products employing mammalian tissue cultures is increasing rapidly as new advances are made in diagnostic procedures and therapeutic treatments. However, current large-scale cell production methods are not economical or reproducible enough to efficiently meet this demand.
Culturing and maintenance of biological materials is a complex science requiring precise balancing of such environmental parameters as nutrients, growth factors, temperature, pH, and the partial pressures of respiratory gases. In addition, it is important to guard against contamination of the culture by bacteria, fungi, their spores, yeast, and viruses. In the past 50 years, a great deal of cell culture research has been conducted on the effect of different media compositions on cell growth and viability. However, research on basic environmental requirements has remained virtually unchanged. Updating culture techniques is a necessary complement to recent advances in cell biology and culture media refinement.
Large scale tissue culture is traditionally carried out in capped roller bottles using bicarbonate buffer systems. The maintenance of adequate oxygen and carbon dioxide levels is a basic requirement for culturing cells in vitro. Appropriate gas exchange is, therefore, of primary concern in tissue culture methodology. Generally, commercial scale roller bottle cell culture is performed in large, heated rooms (approximately 37.degree. C.), in which it is not practical to maintain high CO.sub.2 levels. A typical roller bottle has a volume of approximately one to two liters (490 to 1080 cm.sup.2 surface area), and contains 100 to 300 ml of growth media. It is housed on a roller bottle rack at a 5.degree. incline and rotated at 1-2 revolutions per minute. To obtain the appropriate CO.sub.2 levels, the growth medium is saturated with CO.sub.2 prior to capping the bottles. Dissolved CO.sub.2 from the media diffuses into the bottle atmosphere until equilibrium is reached. This arrangement provides adequate CO.sub.2 partial pressure and proper buffer pH for all phases of cell growth.
As the cells multiply and begin to respire, the concentration of CO.sub.2 in the media and in the atmosphere within the roller bottle begins to rise, causing the media pH to decrease. If the roller bottle remains tightly capped, the pH decreases below the optimum level, and adversely affects the cells. Consequently, roller bottle caps must be partially unscrewed to permit the reduction of the partial pressure of carbon dioxide. Failure to loosen the roller bottle cap causes an increase of the partial pressure of carbon dioxide in the culture environment, which results in suboptimal cell growth or cell death.
Alternatively, if the bottle is completely uncapped or too loosely capped, the dissolved CO.sub.2 in the media will quickly escape and the pH will increase above the optimal level. Such a rise in pH also has a detrimental affect on the cells. Additionally, caps that are too loose permit airborne microbes to enter the roller bottle or media to leak into the threads of the cap, thereby contaminating the culture and destroying its utility.
For example, Mycoplasma contamination constitutes a hazard to cell cultures in terms of the interpretation of data derived from contaminated cells. Mycoplasmas interfere with the growth rate of cultured cells, inhibit or stimulate lymphocyte transformation, induce chromosomal aberrations and alter host cell metabolism and ribosomal RNA and enzyme activities. Stanbridge, E. J. and Doersen, C., in Mycoplasma Infection of Cell Cultures. eds. G. J. McGarrity, D. G. Murphy and W. W. Nichols. 119-212 (Plenum Press. New York, 1978). Control of Mycoplasma contamination is especially important to successful clinical use of cell culture.
Cells are grown in tissue culture flasks in incubators heated to 37.degree. C. with a 5 to 10% CO.sub.2 atmosphere. The covering on the flask permits the equilibration of the media with the incubator environment.
Use of ultra-thin dense skin gas-controlling membrane caps on tissue culture flasks and roller bottles would aid in contamination control and greatly reduce or even eliminate the need for antibiotics in the culture media. This benefit would be especially significant when growing cells for therapeutic purposes. Cultures will not, for example, have penicillin present to elicit a reaction from people allergic to penicillin.
Suboptimal cellular growth caused by improper gas exchange and loss of cultures through cellular death or contamination is costly to the scientific, biomedical, and biotechnical communities in both time and materials. Improvements in cell and tissue culture technology that insure proper respiratory gas exchange and decrease the incidence of culture contamination would increase cell culture efficiency and reduce the costs of these valuable diagnostic and therapeutic materials.
In addition, improvements in the control of the partial pressures of respiratory gases would increase reproducibility and enhance growth rates and yields of in vitro cell cultures by optimizing the CO.sub.2 and O.sub.2 partial pressures in tissue culture containers, maximizing cell growth throughout the growth cycle of the cells and increasing cell growth rates and product yields.
It is therefore an object of the present invention to provide a membrane capable of limiting the exchange of small kinetic diameter gases in cell and tissue culture applications.
It is another object of the present invention to provide a membrane capable of acting as a microbial barrier to bacteria, fungi, their spores, yeast, and viruses.
It is yet another object of the present invention to provide a membrane that is of sufficient strength for use in tissue culture applications, that can be handled and sterilized using standard techniques.
It is a still further object of the present invention to provide caps or covers for culture containers incorporating membranes useful for culturing cells and tissues.